Project Summary. Knowledge of the effects of inflammation on the regenerative functions of periodontal ligament (PDL) cells is incomplete. This limits the development of techniques for periodontal regeneration that will maintain functional tooth support over the long term. Periodontal regeneration includes multiple cellular processes and a less understood component of these processes is PDL cell contractility. Cellular contractile forces are critical to the alignment of collagen fibrils that strengthen periodontal tissue and maintain its functional integrity. The long-term goal of this research is to identify mechanisms regulating PDL cell mechanics that can be used as clinical tools for regenerating and maintaining the architecture and function of the periodontal complex over time. Thus, the objective of this proposal is to demonstrate links between mechanisms regulating PDL cell contractile forces in proinflammatory microenvironments with PDL architecture and tissue mechanics. The central hypothesis of this proposal is that the inflammatory microenvironment regulates PDL cell contractile forces with effects on PDL tissue architecture and mechanics. This hypothesis will be tested in Specific Aim 1 through identification of mechanisms that regulate in vitro PDL cell contractile forces within proinflammatory microenvironments at the single-cell level. Western blots will be used to determine effects of tumor necrosis factor alpha (TNF?) and hyaluronan oligosaccharide (oHA) on signaling pathways that generate cellular contractile force, such as the Rho/Rock pathway. In order to link the inflammatory environment and cell signaling with contractility, cellular traction forces will be measured with and without inflammatory mediators and signaling pathway inhibitors. In Specific Aim 2, three-dimensional PDL constructs will be developed to link the signaling pathways that regulate tissue-level contractility with matrix architecture and stiffness. Engineered PDL constructs will be developed using PDL cells and collagen and in situ forces will be measured. PDL constructs will be treated with stimulants and inhibitors of the Rho/Rock pathway under conditions that model periodontal homeostasis and inflammation. The successful completion of these aims will contribute to the development of clinical techniques for maintaining the PDL or regenerated tissues in a proinflammatory environment. Future research will expand this model to include cementum-like tissue and bone; thus, this pilot study is an initial step toward the future goal of regenerating the periodontal complex and maintaining its functional integrity over the long-term.